Project Summary Idiopathic Pulmonary Fibrosis (IPF) has a prognosis worse than most cancers, with only 20-30 percent of patients surviving 5 years after diagnosis. The pathophysiology in IPF relates to dysregulated repair after injury to the alveolo-capillary though the mechanisms that link alveolar capillary membrane injury with fibroblast activation and disordered repair are incompletely understood. In a recent publication we provide clear genetic evidence that alveolar macrophages are key effector cells in the development of fibrosis in mice which was validated in human alveolar macrophages obtained from pulmonary fibrosis patients. We reported that monocytes recruited to the lung during injury and fibrosis express unique markers and change their morphology to resemble alveolar macrophages. Using combined genetic lineage tracing, a targeted genetic strategy and transcriptomic analysis (RNA-Seq) of flow-sorted myeloid populations we showed that ?monocyte-derived alveolar macrophages? and ?tissue-resident alveolar macrophages? play distinct roles in the development of lung fibrosis. Critically, genetically deleting monocyte derived alveolar macrophages independent of tissue resident alveolar macrophages reduced the severity of bleomycin induced fibrosis. Our results are consistent with findings from others who have suggested that the differentiation from monocytes into tissue-resident macrophages is driven by epigenetic changes in response to cues from the local tissue microenvironment. Because the process of monocyte to alveolar macrophage differentiation is specific to the lung, therapies that target this process after a monocyte has been recruited into the lung are likely to avoid the systemic toxicity associated with systemic monocyte depletion. One feature of the bleomycin lung fibrosis model is the spontaneous resolution of fibrosis over 2-3 months, which does not recapitulate IPF in which continuous progression of lung fibrosis is the norm. However, we have and others have shown that the asbestos mouse model of lung fibrosis does demonstrate progressive fibrosis and thus may more accurately recapitulate human IPF. In order to provide a more compelling rationale for targeting monocyte-macrophage differentiation as a therapeutic target for IPF, we plan to address three important questions raised by our data in our renewal application. First, do monocyte-derived alveolar macrophages play a similar role in asbestos mouse models of non-resolving lung fibrosis? We have already generated preliminary data supporting this hypothesis. As part of these experiments, we will perform single cell transcriptomics (DROP-Seq) to determine whether the expression of pro-fibrotic genes in monocyte-derived alveolar macrophages is attributable to a subpopulation of cells. Second, can the deletion of monocyte-derived alveolar macrophages promote the resolution of fibrosis after it is established? Third, can we use our transcriptomic data to inform strategies to target monocyte-derived alveolar macrophages to ameliorate fibrosis? Our data strongly suggest that lipid metabolism is essential for the differentiation of monocyte-derived alveolar macrophages. We will interrupt this pathway through alveolar macrophage-targeted deletion of the fatty acid synthetase gene (FASN) and pharmacologic inhibition of fatty acid synthesis to examine the effects on monocyte-derived alveolar macrophage differentiation and gene expression during lung fibrosis. In order to establish biological relevance to our in vivo work we will also collect alveolar macrophages from IPF patients and examine transcriptomic differences compared to AM from controls. Specific Aim 1: To determine whether monocyte derived macrophages drive lung fibrosis in non-resolving lung fibrosis models. Specific Aim 2: To determine whether deletion of monocyte-derived macrophages resolves lung fibrosis in the non-resolving lung fibrosis models. Specific Aim 3: To determine whether inhibiting lipid biosynthesis in monocyte-derived macrophages prevents lung fibrosis in the bleomycin and non-resolving lung fibrosis models.